Method of treating scars and β-catenin-mediated disorders using Nefopam compounds

ABSTRACT

Methods of treating disorders mediated by β-catenin comprising administration of Nefopam compounds are provided In particular, the treatment of disorders arising from excessive or undesired expression of β-catenin is contemplated Said compounds include Nefopam, analogues thereof, prodaigs thereof, and salts and solvates of Nefopam The β-catenin mediated disorders are preferably selected from the group consisting of fibroproliferative disorders (such as scars, aggressive fibramtoses, and fibroses) and cancer (such as colon cancer, melanoma, liver cancer, ovarian cancer, endometrial cancer, medullobalstoma pilomatricomas, and prostate cancer)

FIELD OF THE INVENTION

The present invention generally relates to the treatment of scars andβ-catenin-mediated disorders.

BACKGROUND OF THE INVENTION

Fibroproliferative processes are a group of disorders characterized byan excessive proliferation of mesenchymal fibroblast-like spindle cells.They range from hypertrophic wounds to the development of neoplasms suchas aggressive fibromatosis (AF).

During wound healing, several cell types and signaling pathways areactivated to reconstitute the epithelial and dermis layers of the skin.Following cutaneous injury, three sequentially distinct but overlappingprocesses are initiated: inflammatory, proliferation, and remodeling.During the proliferative phase, mesenchymal fibroblast-like cellsaccumulate in the dermal component of the skin while the epithelial cellbarrier layer is reformed (Singer 1999, Martin 1997, McClain 1996).β-catenin has been shown to mediate epithelial and mesenchymal cellactivity, whereby it is able to increase proliferation anddifferentiation in dermal mesenchymal cells and decrease migration inepithelial keratinocytes (Cheon 2002). Mouse models have demonstratedthat β-catenin can modulate the resulting wound size, where inducedlevels of β-catenin by lithium treatment result in wound healing with alarger size (Cheon 2006). Also, a transgenic mouse in which stabilizedβ-catenin is expressed in mesenchymal cells, has been generated, undercontrol of a tetracycline-regulated promoter. Wounded mice healed withhyperplastic cutaneous wounds compared to wildtype control mice (Cheon2002). This demonstrates the importance of β-catenin in mesenchymalcells and its crucial role in wound healing.

Another fibroproliferative disorder mediated by β-catenin is aggressivefibromatosis (AF), also called desmoid tumour. AF is a locally invasivesoft tissue tumour comprised of mesenchymal fibroblast-like spindlecells. AF occurs as either a sporadic lesion or a familial syndrome,such as familial adenomatous polyposis (FAP). B-catenin stabilization isa universal occurrence in AF, as demonstrated by elevated β-cateninlevels and increase β-catenin-mediated transcriptional activity.Furthermore, β-catenin stabilization is sufficient to cause AF as shownusing a transgenic mouse model that over-expresses the stabilized formof β-catenin (Cheon 2002). This suggests a crucial role β-catenin playsin fibroproliferative disorders and its importance in mesenchymal cells.

In addition to a role for β-catenin in fibroproliferative disorders, anumber of studies have demonstrated deregulated β-catenin expression isan important event in the genesis of a number of malignancies, such ascolon cancer, melanoma, hepatocellular carcinoma, ovarian cancer,endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer.β-catenin mutations appear to be a crucial step in the progression of asubset of these cancers, suggesting an important role in the control ofcellular proliferation or cell death (as described in Polakis P. Themany ways of Wnt in cancer. Curr Opin Genet Dev. 2007 February;17(1):45-51).

In view of foregoing, it is desirable to develop novel methods effectiveto treat conditions and disorders that may be associated with β-catenin.

SUMMARY OF THE INVENTION

It has now been found that Nefopam, and analogues thereof, are useful totreat disorders mediated by β-catenin, such as fibroproliferativedisorders, as well as treating scar tissue.

Accordingly, in one aspect of the invention, a method of treating aβ-catenin-mediated disorder or condition in a mammal is providedcomprising administering Nefopam, or a functionally equivalent analogue,prodrug, salt or solvate thereof, to the mammal.

In another aspect of the invention, a method of treating scar tissue orreducing scar tissue formation comprising administration to the tissue atherapeutically effective amount of Nefopam, or a pharmaceuticallyacceptable analogue, salt, solvate or prodrug thereof.

In an alternative aspect, an article of manufacture is providedcomprising packaging and a composition comprising Nefopam or afunctionally equivalent analogue, salt, solvate or prodrug thereof.

In another aspect, the novel use of Nefopam or a functionally equivalentanalogue, salt, solvate or prodrug thereof, is provided for thepreparation of a medicament for the treatment of a β-catenin-mediateddisorder or condition, or for the treatment of scar tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in further detail withreference to the following Figures:

FIG. 1A is a bar graph indicating cell viability in cultured normalfibroblast cells and cultured cells derived from two hyperplastic woundsfollowing treatment with DMSO (control) or Nefopam using the SRB assay.Percent cell survival is given as a mean and 95% confidence interval.There is significant decline in the percent of cells surviving incultures treated with Nefopam compared to DMSO control hyperplasticwound cell cultures, however, cell survival rates in normal fibroblastcultures remained relatively unchanged (asterisk indicates significancecompared to normal fibroblast cultures).

FIG. 1B is a western blot analysis of β-catenin levels in hyperplasticwound cell cultures. Nefopam treatment was shown to substantially reduceβ-catenin protein levels compared to DMSO treated controls.

FIG. 2 is a graph comparing the number of aggressive fibromatosis (AF)tumours formed in male Apc+/Apc1638N mice left untreated or treated withNefopam or DMSO control and illustrating the number ofepithelial-derived polyps in the upper gastrointestinal tract under thesame treatment. 1) No Treatment (n=11), 2) 0.1% DMSO (n=10), and 3)Nefopam at 40 mg/kg body weight (n=10).

FIG. 3A is a western blot of β-catenin protein levels (92 kDa) inextracts from primary cell cultures derived from human aggressivefibromatosis (AF) tumours (n=5) following treatment for 5 days with oneof 0.1% DMSO (control) or Nefopam. β-catenin protein levels were alsodetermined in primary fibroblast cell cultures incubated with Wnt3a withor without Nefopam. Experiments were performed in triplicate. Actinexpression is shown as a lysate loading control.

FIG. 3B is a graph of densitometry analysis of protein level datashowing a nearly 5-fold decrease in total β-catenin protein levels incell cultures derived from human AF tumours treated with 0.1% DMSO(control) or Nefopam. Means and 95% confidence intervals are shown.Statistically significant differences (p<0.05) compared to the controlare indicated by an asterisk.

FIG. 4A is a graph showing the means and 95% confidence intervals ofcell viability of primary cells derived from human AF tumours treatedwith DMSO (n=5) or Nefopam (n=5) for 5 days. Cell viability was measuredby staining cells with Trypan Blue Dye and counting both live (clear)and dead (blue) cells. Nefopam significantly decreased the number oflive cells while the number of dead cells did not change. Statisticallysignificant differences (p<0.05) compared to controls are indicated withan asterisk

FIG. 4B is a graph showing percent BrdU-positive/DAPI-positive cellscompared to total DAPI-positive cells as a measure of proliferation incultures of primary cells derived from human aggressive fibromatosistumours (n=2) treated with DMSO or Nefopam in triplicate for 5 days.Nefopam significantly reduces the incorporation of BrdU into cells. Themeans and 95% confidence intervals are shown. Statistically significantdifferences (p<0.05) compared to the control are indicated by asterisk.

FIG. 5A shows western blot analysis of lysates extracted fromimmortalized human fibroblast cells. A significant decrease in totalβ-catenin protein levels in cells treated with Nefopam compared to cellstreated with DMSO was observed. GAPDH expression is shown as a lysateloading control.

FIG. 5B is a graph of densitometry data corresponding to western blotdata of FIG. 5A.

FIG. 6A is a western blot analysis of β-catenin protein levels in cellcultures from Tcf mice wounds 14 days post-wounding. GAPDH expression isshown as lysate loading control.

FIG. 6B is a graph of normal scar size in mice subjected to fullthickness circular wounds following treatment with either Nefopamformulated with a carrier (Nefopam) or carrier alone (control)administered systemically as 40 mg/kg daily for two weeks. The graphshows the mean and 95% confidence interval for the diameter of thesurface area of a cutaneous wound generated using a 4 mm biopsy punch.The diameter of the wound is significantly smaller following Nefopamtreatment compared to control treatment (asterisk indicates asignificant difference).

FIG. 7 is a line graph indicating relative β-catenin protein levels overtime (measured in weeks) during normal wound healing (normal) and inhyperplastic wounds (hyperplastic) compared to unwounded tissue. Thenormal pattern of rise and fall of β-catenin protein levels duringnormal wound healing is deregulated in hyperplastic wounds, whichexhibit a significantly prolonged duration of elevated β-catenin proteinlevels.

FIG. 8 is a graph of the mean and 95% confidence interval for thediameter of the surface area of cutaneous hyperplastic scars four weekspost-wounding. 4 mm diameter full thickness circular wounds weregenerated using a biopsy punch. Wound diameter is given in mm. Anasterisk indicates statistically significant differences in scar sizenoted when compared to treatment with TGF-β (p<0.01), where TGF-βinjection at the time of wounding is known to cause hyperplastic scarsof increased size.

FIG. 9 is a graph of varying concentration Nefopam topical formulationsin three different carriers: carboxymethylcellulose (CMC), petrolatum,and hypromellose. The three carriers were tested in vivo in a mousemodel to determine the formulation most effective in delivering Nefopamthrough the skin. Petrolatum-based carrier formulations demonstratedenhanced Nefopam release properties as determined by measurement ofNefopam levels in the skin and serum.

FIG. 10 is a graph of relative scar surface area measured in arbitraryunits (the scar size upon wounding is considered as 100 arbitraryunits). Full thickness puncture wounds 4 mm in diameter were treatedtopically with either carrier control cream or 1% Nefopam creamformulated in petrolatum carrier twice daily for 14 days. The datarepresent an average of 10 wounds per treatment with standard deviation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method of treating a β-catenin-mediated disorder in a mammal isprovided comprising administering Nefopam or a functionally equivalentanalogue thereof to the mammal.

As used herein, the term “β-catenin-mediated disorder or condition”refers to disorders or conditions characterized by the accumulation offibrous tissue (“fibrosis”) including, but not limited to,fibroproliferative disorders such as dermal scars includinghypertrophic, hyperplastic and keloid scars, either in formation oralready formed, and aggressive fibromatoses e.g. sporadic lesion or afamilial syndrome such as familial adenomatous polyposis (FAP), liverfibrosis, lung fibrosis (e.g., silicosis, asbestosis), kidney fibrosis(including diabetic nephropathy), glomerulosclerosis, Lederhose diseaseand Dupuytren's contracture (DC), as well as malignancies, such as coloncancer, colorectal cancer, melanoma, hepatocellular carcinoma, ovariancancer, endometrial cancer, medulloblastoma pilomatricomas, and prostatecancer.

The term “Nefopam” refers to5-methyl-1-phenyl-1,3,4,6-tetrahydro-2,5-benzoxazocine andpharmaceutically acceptable functionally equivalent analogues, prodrugs,salts and solvates thereof. The term “functionally equivalent”, as itused with respect to analogues, prodrugs, salts and solvates of Nefopam,refers to the ability of the selected compound to modulate β-catenin.The extent to which the selected compound may modulate β-catenin mayvary from compound to compound.

The term “analogue” as used herein refers to compounds having thefollowing general formula (1),

wherein R₁ is H, C₁-C₆ alkyl optionally substituted with F orC₃-C₆cycloalkyl or C₂-C₄alkenyl; A is O, CH₂ or S(O)_(n) where n is 0-2;one of W, X, Y and Z is N, CH or CR₃ and the others are CH; R₂ is C₅-C₆heteroaryl, C₅-C₁₀ cycloalkyl or cycloalkenyl optionally containing oneor more heteroatoms selected from O, N and S(O)_(n) where n is 0-2, andoptionally substituted with R₃; or a phenyl group optionally substitutedin one or more positions with one or more substituents independentlyselected from halogen, CN, CF₃, C₁-C₆ alkyl and OR₁, or the phenyl groupis fused to a five or six membered ring which may be carbocyclic,heterocyclic (containing 1-2 heteroatoms selected from O, N and S),aromatic or heteroaromatic (containing 1-2 heteroatoms selected from Oand N); R₃ is selected from halogen; CF₃; CN; OR₅; SO₂N(R₅)₂; COR₅;CO₂R₅; CON(R₅)₂; NR₁COR₄; NR₁SO₂R₄; NR₁CO₂R₄; NR₁CON(R₅)₂; OC₁-C₆ alkylsubstituted with R₃; C₁-C₆ alkyl optionally substituted withunsubstituted R₃; C₃-C₆cycloalkyl optionally substituted withunsubstituted R₃; C₂-C₆alkenyl optionally substituted with unsubstitutedR₃; C₂-C₆alkynyl optionally substituted with unsubstituted R₃; aryloptionally substituted with unsubstituted R₃; and five or six memberedaromatic heterocycles containing 1-4 heteroatoms selected from N and O;R₄ is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₅ is H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₆cycloalkyl, aryl or heteroaryl and is the same as or different toanother R₅; or a pharmaceutically acceptable salt thereof; wherein R₁ isH, C₁-C₆ alkyl, optionally substituted with F or C₃-C₆cycloalkyl orC₂-C₆ alkenyl; R₂ and R₃ are the same or different and are H, a halogen,CN, CF₃, C₁-C₆ alkyl or OR₁, or R₂ and R₃ form a five or six memberedring which may be carbocyclic, heterocyclic (containing 1-2 heteroatomstaken from O, N and S), aromatic or heteroaromatic (containing 1-2heteroatoms taken from O and N); one of W, X, Y and Z is N, or CR₄ andthe others are each CH; R₄ is a halogen atom, CF₃, CN, OR₇, SO₂N(R₆)₂,COR₆, CO₂R₆, CON(R₆)₂, NR₁COR₅, NR₁SO₂R₅, NR₁CO₂R₅, NR₁CON(R₆)₂, OC₁-C₆alkyl optionally substituted with R₄, C₁-C₆ alkyl optionally substitutedwith R₄, C₃-C₆cycloalkyl optionally substituted with R₄, C₂-C₆alkenyloptionally substituted with R₄, C₂-C₆alkynyl optionally substituted withR₄, aryl optionally substituted with R₄, or a five or six memberedaromatic heterocycle containing 1-4 heteroatoms selected from N and O,linked either through carbon or nitrogen; R₅ is C₁-C₆ alkyl,C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl or heteroaryl; each R₆(which may be the same or different) is H, C₁-C₆ alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl or heteroaryl; and R₇ is aryl orheteroaryl; or a pharmaceutically acceptable salt thereof; wherein R₁ isH, C₁-C₆ alkyl optionally substituted with F or C₃-C₆cycloalkyl orC₂-C₄alkenyl; A is O, CH₂ or S(O)_(n) where n is 0-2; one of W, X, Y andZ is N, CH or CR₃ and the others are CH; R₂ is C₅-C₆heteroaryl, C₅-C₁₀cycloalkyl or cycloalkenyl optionally containing one or more heteroatomsselected from O, N and S(O)_(n) where n is 0-2, and optionallysubstituted with R₃; or a phenyl group optionally substituted in one ormore positions with one or more substituents independently selected fromhalogen, CN, CF₃, C₁-C₆ alkyl and OR₁, or the phenyl group is fused to afive or six membered ring which may be carbocyclic, heterocyclic(containing 1-2 heteroatoms selected from O, N and S), aromatic orheteroaromatic (containing 1-2 heteroatoms selected from O and N); R₃ isselected from halogen; CF₃; CN; OR₅; SO₂N(R₅)₂; COR₅; CO₂R₅; CON(R₅)₂;NR₁COR₄; NR₁SO₂R₄; NR₁CO₂R₄; NR₁CON(R₅)₂; OC₁-C₆ alkyl substituted withR₃; C₁-C₆ alkyl optionally substituted with unsubstituted R₃;C₃-C₆cycloalkyl optionally substituted with unsubstituted R₃;C₂-C₆alkenyl optionally substituted with unsubstituted R₃; C₂-C₆alkynyloptionally substituted with unsubstituted R₃; aryl optionallysubstituted with unsubstituted R₃; and five or six membered aromaticheterocycles containing 1-4 heteroatoms selected from N and O; R₄ isC₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₅ is H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₆cycloalkyl, aryl or heteroaryl and is the same as or different toanother R₅; or a pharmaceutically acceptable salt thereof; wherein R₁ isH, C₁-C₆ alkyl optionally substituted with F or C₃-C₆cycloalkyl orC₂-C₄alkenyl; A is O, CH₂ or S(O)_(n) where n is 0-2; one of W, X, Y andZ is N, CH or CR₃ and the others are CH; R₂ is C₅-C₆ heteroaryl, C₅-C₁₀cycloalkyl or cycloalkenyl optionally containing one or more heteroatomsselected from O, N and S(O)_(n) where n is 0-2, and optionallysubstituted with R₃; or a phenyl group optionally substituted in one ormore positions with one or more substituents independently selected fromhalogen, CN, CF₃, C₁-C₆ alkyl and OR₁, or the phenyl group is fused to afive or six membered ring which may be carbocyclic, heterocyclic(containing 1-2 heteroatoms selected from O, N and S), aromatic orheteroaromatic (containing 1-2 heteroatoms selected from O and N); R₃ isselected from halogen; CF₃; CN; OR₅; SO₂N(R₅)₂; COR₅; CO₂R₅; CON(R₅)₂;NR₁COR₄; NR₁SO₂; NR₁CO₂R₄; NR₁CON(R₅)₂; OC₁-C₆ alkyl substituted withR₃; C₁-C₆ alkyl optionally substituted with unsubstituted R₃;C₃-C₆cycloalkyl optionally substituted with unsubstituted R₃;C₂-C₆alkenyl optionally substituted with unsubstituted R₃; C₂-C₆alkynyloptionally substituted with unsubstituted R₃; aryl optionallysubstituted with unsubstituted R₃; and five or six membered aromaticheterocycles containing 1-4 heteroatoms selected from N and O; R₄ isC₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₅ is H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₆cycloalkyl, aryl or heteroaryl and is the same as or different toanother R₅; or a pharmaceutically acceptable salt thereof; or wherein:R₁ is H, C₁-C₆ alkyl, optionally substituted with F or C₃-C₆ cycloalkylor C₂-C₄ alkenyl; R₂ and R₃ are the same or different and are each H,halogen, CN, CF₃, C₁-C₆ alkyl or OR₁, or R₂ and R₃ may form a five orsix membered ring which may be carbocyclic, heterocyclic (containing 1-2heteroatoms taken from O, N and S), aromatic or heteroaromatic(containing 1-2 heteroatoms taken from O and N); and one of W, X, Y andZ is N, CH or CR₄ and the others are CH; R₄ is halogen; CF₃; CN; OR₇;SO₂N(R₆)₂ (where each R₆ is the same or different); COR₆; CO₂R₆;CON(R₆)₂ (where R₆ is the same or different); NR₁COR₅; NR₁SO₂R₅;NR₁CO₂R₅; NR₁CON(R₆)₂ (where each R₆ is the same or different), OC₁-C₆alkyl substituted with unsubstituted R₄, C₁-C₆ alkyl optionallysubstituted with unsubstituted R₄, C₃-C₆cycloalkyl optionallysubstituted with unsubstituted R₄, C₂-C₆alkenyl optionally substitutedwith unsubstituted R₄, C₂-C₆alkynyl optionally substituted withunsubstituted R₄ and aryl optionally substituted with unsubstituted R₄,or R₄ is a five or six membered aromatic heterocycle containing 1-4heteroatoms taken from N and O; R₅ is C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl or heteroaryl; R₆ can be H,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₆cycloalkyl, aryl orheteroaryl; and R₇ is aryl or heteroaryl; or a pharmaceuticallyacceptable salt thereof. Additional analogues of Nefopam are describedin WO2004/056788, WO2005/103019 and US 2006/0019940, the contents ofwhich are incorporated herein by reference. Nefopam and analoguesthereof may be made using chemical synthetic methods well-known to thosein the art. In addition, Nefopam is commercially available.

The term “prodrug” refers to a compound (e.g. a drug precursor) that istransformed in vivo to yield a compound having the structure of Nefopamor an pharmaceutically acceptable analogue, salt, hydrate or solvatethereof. The transformation may occur by various mechanisms (e.g., bymetabolic or chemical processes), such as, for example, throughhydrolysis in blood. The term “salt(s)”, as employed herein, denotesacidic salts formed with inorganic and/or organic acids, as well asbasic salts formed with inorganic and/or organic bases. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful. A “solvate” is formedby admixture of Nefopam or an analogue thereof in a solvent which ispreferably pharmaceutically acceptable.

The present method encompasses the treatment of a β-catenin-mediateddisorder in a mammal. The terms “treat”, “treating” and “treatment” areused broadly herein to denote methods that favorably alter the targeteddisorder, including those that moderate or reverse the progression of,or reduce the severity of, the disorder. The term “mammal” is usedherein to encompass both human and non-human mammals.

A method of treating dermal scars, including scars resulting from cuts,scrapes, infection, acne, burns, surgery, etc., hypertrophic,hyperplastic, keloid, scars involving mesenchymal andmesenchymal-derived cells, any of which may be B-catenin-mediated ornot, is also provided. The method comprises administering to the targetsite a therapeutically effective amount of a Nefopam compound. Themethod of treating scars, in formation or already formed, includesreducing the size of the scar (for example, by at least about 5-10%,preferably by at least about 20%, and more preferably by at least about25% or more) or prevalence of the scar (e.g. elevation of the scar,redness, etc.) and thereby improving the appearance thereof. In thisregard, as one of skill will appreciate, a scar assessment scale, e.g.the Manchester Scale, may be used to assess the improvement of a givenscar. The Manchester Scale assesses colour compared with surroundingskin, matte or shiny appearance, contour (flush with surrounding skin toscar/keloid), texture (normal to hard), margins (distinct or not), sizeand number (single or multiple) (Disability & Rehabilitation, 2009, Vol.31, No. 25: Pages 2055-2063; International Journal of Lower ExtremityWounds December 2007 6: 249-253).

Thus, Nefopam compounds may be utilized in a cosmetic treatment toreduce scar tissue and improve the aesthetics of the scar andsurrounding area, and may provide additional cosmetic features, e.g.anti-wrinkling effects.

In another embodiment, a method of treating tumours is provided. Tumourtreatment includes inhibiting tumour initiation and tumour cellproliferation. The method is useful to treat tumours resulting fromderegulated β-catenin expression such as aggressive fibromatosis, aswell as tumours resulting from various cancers such as colon cancer,melanoma, hepatocellular cancer, ovarian cancer, endometrial cancer andprostate cancer. The method comprises administering to a mammal in needof treatment, i.e. a mammal having a tumour, an effective amount ofNefopam, an analogue thereof, or a pharmaceutically acceptable salt,solvate or prodrug thereof.

While not wishing to be bound by any particular theory, treatment inaccordance with the present invention may be effected by the regulationor modulation of β-catenin expression, at the nucleic acid level, or theregulation or modulation of β-catenin activity, at the protein level.

Therapeutically effective dosages of Nefopam are administered to amammal in accordance with the invention. The term “therapeuticallyeffective” as it is used herein with respect to dosages refers to adosage that is effective to treat a β-catenin-mediated disorder withoutcausing unacceptable adverse side effects. The term “administered”refers to any appropriate means of providing Nefopam to a mammal, andwill depend on the dosage form being used as will be described. Forexample, the dosage may be administered orally, by injection, mucosallyand topically as will be described in more detail.

Therapeutically effective dosages according to the method, thus, are inthe range of about 0.0001 to about 1500 mg, for example, in a range ofabout 0.0001-100 mg. However, as one of skill in the art willappreciate, the effective therapeutic dosage of Nefopam, or analoguesthereof, will vary depending many factors, including but not limited to,the type of disorder to be treated, the nature and severity of thedisorder, the mammal to be treated, the symptoms of the mammal beingtreated, the compound used for the treatment, and the route ofadministration.

Nefopam may be administered in accordance with methods of the inventionalone or in a composition combined with a pharmaceutically acceptableadjuvant or carrier. The expression “pharmaceutically acceptable” meansacceptable for use in the pharmaceutical arts, i.e. not beingunacceptably toxic, or otherwise unsuitable for administration to amammal. Examples of pharmaceutically acceptable adjuvants include, butare not limited to, diluents, excipients and the like. Reference may bemade to “Remington's: The Science and Practice of Pharmacy”, 21st Ed.,Lippincott Williams & Wilkins, 2005, for guidance on drug formulationsgenerally. The selection of adjuvant depends on the intended mode ofadministration of the composition. In one embodiment of the invention,the compounds are formulated for administration by infusion, or byinjection either subcutaneously or intravenously, and are accordinglyutilized as aqueous solutions in sterile and pyrogen-free form andoptionally buffered or made isotonic. Thus, the compounds may beadministered in distilled water or, more desirably, in saline,phosphate-buffered saline or 5% dextrose solution. Compositions for oraladministration via tablet, capsule, lozenge, solution or suspension inan aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquidemulsion, an elixir or syrup are prepared using adjuvants includingsugars, such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and derivatives thereof, includingsodium carboxymethylcellulose, ethylcellulose and cellulose acetates;powdered tragancanth; malt; gelatin; talc; stearic acids; magnesiumstearate; calcium sulfate; vegetable oils, such as peanut oils, cottonseed oil, sesame oil, olive oil and corn oil; polyols such as propyleneglycol, glycerine, sorbital, mannitol and polyethylene glycol; agar;alginic acids; water; isotonic saline and phosphate buffer solutions.Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers,tableting agents, disintegrating agents, anti-oxidants, preservatives,colouring agents and flavouring agents may also be present. In anotherembodiment, the composition may be formulated for application topicallyas a cream, lotion or ointment. For such topical application, thecomposition may include an appropriate base such as a triglyceride base.Such creams, lotions and ointments may also contain a surface activeagent and other cosmetic additives such as skin softeners and the likeas well as fragrance. Aerosol formulations, for example, for nasaldelivery, may also be prepared in which suitable propellant adjuvantsare used. Compositions of the present invention may also be administeredas a bolus, electuary, or paste. Compositions for mucosal administrationare also encompassed, including oral, nasal, rectal or vaginaladministration for the treatment of infections which affect these areas.Such compositions generally include one or more suitable non-irritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax, a salicylate or other suitablecarriers. Other adjuvants may also be added to the compositionregardless of how it is to be administered which, for example, may aidto extend the shelf-life thereof.

In accordance with the present method, a Nefopam compound may beadministered in a convenient manner by any of a number of routesincluding but not limited to oral, subcutaneous, intravenous,intraperitoneal, intranasal, enteral, topical, sublingual,intramuscular, intra-arterial, intramedullary, intrathecal, inhalation,ocular, transdermal, vaginal or rectal means. Nefopam compounds may alsobe administered to cells in ex vivo treatment protocols. Depending onthe route of administration, Nefopam compounds may be coated or encasedin a protective material to prevent degradation by, e.g. enzymes, acidsor other conditions that may affect the therapeutic activity thereof.

In one embodiment, Nefopam, or an analogue thereof, may be topicallyapplied to a target site, e.g. a scar in formation or already formed,affixed to a biocompatible device, polymer or other matrix, e.g. such asa bandage, dressing, polymer mesh, implant, device or other cosmeticallyrelated item. Dermal fibroblasts/keratinocytes bioengineered to expressa Nefopam compound may also be applied to a target site. A suitablematrix or polymer mesh, e.g. artificial or non-artificial skin grafts,may alternatively be impregnated with a Nefopam compound for applicationto a target site to permit slow-release of the compound for continuoustreatment of the site over a period of time.

The present Nefopam compounds may be administered in a controlledrelease formulation using well-established methods including, forexample, by dissolution or diffusion-controlled monolithic devices,beaded encapsulated systems, osmotically controlled systems, andmodified film coating systems incorporating suitable polymeric andnon-polymeric hydrophilic and hydrophobic materials. Suitablecontrolled-release formulations may include hydrophilic materialscomprising, but not limited to, acrylic or methacrylic polymers orcopolymers, alkylvinyl polymers, celluloses, hydroxyalkyl celluloses,carboxyalkyl celluloses, polysaccharides, alginates, pectins, starchesand derivatives, natural and synthetic gums, polycarbophil, chitosans.Suitable hydrophobic materials comprise, but are not limited to,hydrophobic polymers, waxes, fats, long-chained fatty acids, theircorresponding esters, their corresponding ethers, and their mixtures.

In another embodiment, Nefopam compounds may be administered incombination with one or more additional therapeutic agents, includingfor example, an anti-scarring agent; a wound healing agent such as agrowth factor, e.g. epidermal growth factor, bFCF, PDGF; platelets,dermal fibroblasts and keratinocytes; chemotherapeutic agents such as,but not limited to, rapamycin, troglitazone, rosiglitazone, celecoxib,retinoids and iressa. In this regard, Nefopam may be administered in aseparate formulation, or together with an additional therapeutic agentin a combined formulation.

In addition, the present methods may be utilized in a combination withother therapies, for example, in combination with radiation therapy inthe treatment of malignancies, or in combination with laser therapy totreat scar tissue such as normal scars, hyperplastic scar tissue and thelike.

In a further aspect of the invention, an article of manufacture isprovided comprising packaging and a composition comprising Nefopam asdescribed. The packaging is labelled to indicate that the composition issuitable to treat a β-catenin-mediated disorder, or may be labelled toindicated that the composition is suitable to treat scarring, either information or already formed.

The present invention is described by reference to the accompanyingFigures and specific Examples which are not to be construed as limiting.

EXAMPLES

The following materials and methods were used in the examples discussedbelow.

Apc⁺/Apc^(163N) AF mouse model and treatment plan. The generation andphenotype of Apc/Apc1638N mice have been well characterized. These miceharbour a targeted mutation at codon 1638 in the Apc gene as a result ofa neomycin insert in antisense orientation at exon 15. Male mice developan average of 45 AF lesions and 6 gastrointestinal polyps by the age of6 months, while female mice develop significantly fewer numbers of AFlesions. Male Apc/Apc1638N mice were divided into three study groups: NoTreatment (n=11), 0.1% DMSO (n=10), and Nefopam at 40 mg/kg body weight(n=10). Treatment by daily oral gavaging began 2 months afterApc/Apc1638N mice were weaned and continued for 3 months. At autopsy, AFtumours and intestinal polyps were scored macroscopically. AF tumoursand normal tissue were collected for protein extraction and fixed forhistological examination.

Tcf reporter mice and wounding experiments. A Tcf-reporter constructcontaining the lacZ gene downstream of a c-Fos minimal promoter andthree consensus Tcf-binding motifs was constructed. Upon binding ofβ-catenin/Tcf complex to Tcf motifs, the expression of lacZ isactivated. Tcf mice were wounded as described previously: two 4 mmdiameter full-thickness skin wounds were generated using a dermal biopsypunch (Miltex Instrument Company, York, Pa., USA). Wounded Tcf mice wereseparated into two study groups: Control group, which received dailyintraperitoneal injections of Saline; and Nefopam group, which receiveddaily intraperitoneal injections of 40 mg/kg body weight. At 14 dayspost-wounding, wound sizes were examined, and wound tissues werecollected for RNA and protein extraction and fixed for histologicalexamination.

Human AF Tumour and Normal Fascial Tissue Samples.

Samples of human aggressive fibromatosis tumours were obtained at thetime of surgery from the Hospital for Sick Children, Toronto. Tumourtissue and surrounding normal fascial tissue from the same patient wereharvested and processed immediately after surgical excision. Tissueswere cryopreserved and stored in liquid nitrogen vapour.

Cell Culture Studies.

Primary cell cultures from the human AF tumour and normal fascial tissuesamples were established. Monolayer cultures were cultured in DMEMsupplemented with 10% fetal bovine serum and maintained at 37° C. in 5%CO₂. Cells were divided when confluent and experiments were performedbetween the first and fifth passages. Prior to experimental studies,cells were seeded overnight and treatment began the following day (Day0) where cells were treated with vehicle control 0.1% DMSO with orwithout 250 μm Nefopam prepared in DMEM media.

Cell Viability Assay, Proliferation Assay and Apoptosis Assay werePerformed.

Cell viability was measured using the Trypan Blue Dye Exclusion method.Cells were stained with Trypan Blue Dye at a 1:1 ratio, and both live(clear) and dead (blue) cells were accounted for. Proliferation wasmeasured using 5-bromo-2-deoxy-uridine (BrdU) Incorporation assay. AfterBrdU incubation for 12 hours, cells with incorporated BrdU wereidentified using rabbit monoclonal anti-BrdU antibody and horseanti-mouse antibody conjugated to Alkaline Phosphatase. Presence of BrdUwas detected using Alkaline Phosphatase substrate. Percentage ofpositively stained nuclei out of total nuclei was analyzed over 10high-powered fields.

Protein Extraction and Western Blot Analysis.

Tissue samples were washed twice with PBS and lysed with Reporter GeneAssay Lysis Buffer (Roche). Lysates were centrifuged at 16,000×g for 5minutes to remove cell debris and quantified using the BicinchoninicAcid (BCA) Protein Assay (Pierce). Equal amounts of total protein wereseparated by electrophoresis through an SDS-polyacrylamide gel,transferred to a nitrocellulose membrane (Amersham), and immunoblottedovernight at 4° C. with primary antibodies against phosphoGSK3β (Ser 9,rabbit polyclonal, New England Biolabs), β-catenin (mouse monoclonal,Upstate Biotechnology), total GSK3β (mouse monoclonal, TransductionLaboratories), and GAPDH (mouse monoclonal, Upstate Biotechnology).Horseradish Peroxidase (HRP)-tagged secondary antibodies and EnhancedChemiLuminescence (Amersham) were used to detect hybridization.Densitometery was performed using the AlphaEaseFC software (AlphaInnotech). Western blotting was performed in triplicates to ensurereproducibility.

Statistical Analysis.

Data in this work are presented as mean±95% confidence intervals. Allstudies were performed in at least triplicates to ensurereproducibility.

Example 1 Nefopam Treatment Reduces Hyperplastic Wound Cell Viability

Compounds were screened to identify those that meet two criteria: 1)inhibit cell viability of fibroblasts obtained from hyperplastic woundswhich exhibit β-catenin activation; and 2) show little to no effect onnormal dermal fibroblast cultures. The biological relevance of thescreen was considerable since cells used for the screen were obtainedfrom patients with hyperplastic wounds as well as healthy tissue. Theexperiments were repeated in triplicate within 96 well plates, with eachwell containing 4000 cells treated with between 0.1 1.0, or 10 μM ofcompound or DMSO as a control. The Sulforhodamine B assay (SRB) was usedto measure cell viability. Compounds detected within the initial screenunderwent further testing using a larger pool of samples, from whichNefopam was identified (see FIG. 1A).

β-catenin levels in cell cultures from hyperplastic scars treated withNefopam or control were analyzed using Western blot analysis. It wasobserved that Nefopam substantially reduced the protein level ofβ-catenin in cell cultures from hyperplastic wounds (see FIG. 1B). GAPDHexpression was included as a loading control.

Example 2 Nefopam Decreases the Number of AF Tumours Formed inApc/Apc1638N Mice

It was investigated whether or not Nefopam treatment was able tomodulate the phenotype of AF lesions in vivo. The number of AF tumoursformed in male Apc/Apc1638N mice treated with Nefopam was significantlyreduced compared to the number formed in untreated mice or mice treatedwith 0.1% DMSO at 6 months of age (8.18±1.77 vs 13.2±2.30 or 12.09±1.31,p<0.03, see FIG. 2). There were no significant differences in the numberof epithelial-derived polyps in the upper gastrointestinal tract (seeFIG. 2). This shows that Nefopam inhibits tumour initiation and furtheris specific to mesenchymal cells.

Example 3 Nefopam Decreases β-Catenin Levels in Human AF Tumour Cells

AF tumours are characterized by an increase in (β-catenin levels. Toexamine whether Nefopam has the capacity to modulate β-catenin levels,primary cell cultures derived from several human AF tumours werestudied. Western blot analysis using an antibody against total β-catenindemonstrated a marked decrease in the amount of protein at size 92 kDaconsistent with total β-catenin as a result of Nefopam treatment for 5days, see FIG. 3A. Densitometry analysis showed nearly a 5-fold decreasein total β-catenin levels in human AF tumour cell cultures treated withNefopam compared to those treated with 0.1% DMSO (see FIG. 3B). Actinexpression was determined as a lysate loading control.

Example 4 Nefopam Decreases Cell Viability and Cell Proliferation inHuman AF Tumour Cells

To determine how Nefopam may modify AF cell behaviour, primary cellcultures derived from several human AF tumours were studied. First, theeffects of Nefopam on cell viability in human AF tumours were studied. Asignificantly smaller number of live cells were observed in human AFtumour cell cultures following Nefopam treatment compared to culturestreated with 0.1% DMSO (p<0.05). There were no significant differencesin the number of dead cells counted as a result of Nefopam treatment forthe tumours (p<0.05) (see FIG. 4A).

Upon demonstrating that β-catenin levels are involved in the regulationof the rate of proliferation in mesenchymal cells, the effects ofNefopam on proliferation in primary cell cultures were investigated.Using the BrdU incorporation assay, the percentage of BrdU+/DAPI+ cellscompared to total DAPI+ cells was measured. It was observed thatNefopam-treated human AF tumours contained significantly fewerproliferating cells as determined by BrdU incorporation (p<0.05, seeFIG. 4B).

Together, these results show that Nefopam preferentially inhibits thenumber of viable AF cells by reducing the rate of proliferation.

Example 5 Nefopam Decreases β-Catenin Levels in Primary Human FibroblastCell Cultures

Hyperplastic wounds are characterized by elevated β-catenin levelsduring the proliferative phase. The data described herein show thatNefopam has the capacity to modulate β-catenin levels particularly inmesenchymal-derived cells. To confirm that Nefopam can modulateβ-catenin levels in mesenchymal cells, immortalized human fibroblastcells were treated with Nefopam (see FIG. 5A). Nefopam treatmentresulted in an approximately 4-fold decrease in total β-catenin levelsin primary human fibroblast cell cultures compared to cultures treatedwith 0.1% DMSO as determined by densitometry analysis (p<0.05, see FIG.5B). Additional controls included in the experiments were Wnt3atreatment of cells (known to increase β-catenin expression) and effectsof Nefopam on cells treated with Wnt3a.

Example 6 Systemic Nefopam Decreases β-Catenin Levels and Wound Sizes inTcf Mice

Next, to examine the effects of Nefopam on β-catenin levels during woundhealing, wounded tissue from Tcf mice were studied. Cutaneous woundswere generated using a biopsy punch procedure resulting in a 4 mmdiameter full thickness circular wound. Scale is in mm units. Westernblotting using an antibody against total β-catenin (see FIG. 6A)demonstrated a decrease in β-catenin levels in cells cultured fromwounds derived from Tcf mice treated with Nefopam compared to thecontrol group 14 days post-wounding.

Furthermore, examination of the wounds upon autopsy showedNefopam-treated mice had scars significantly smaller in diametercompared to carrier (saline) treated controls at day 14 post-wounding(asterisk indicates a significant difference, p<0.001) (see FIG. 6B).

Example 7 Systemic Nefopam Reduces Hyperplastic Scar Size Induced byTGF-β

It is known that β-catenin protein levels increase during the earlystages of wound healing then fall through later stages relative tounwounded tissue. The normal rise and fall of β-catenin protein levelsare deregulated during hyperplastic wound healing where significantlyprolonged elevated levels of β-catenin are observed (see FIG. 7).

Following drug screen studies, effects of Nefopam were tested in vivousing mice. Both oral and intraperitoneal administration routes wereevaluated (40 mg/kg body weight, daily; 0.1% DMSO as control). In bothadministration routes, Nefopam was identified in the serum as detectedusing HPLC (data not shown). 4 mm full thickness punch wounds were madein the skin, and Nefopam or control was administered daily afterwounding. To determine if Nefopam is effective in treating hyperplasticscars, a mouse hyperplastic scar model, in which TGF-® is injected priorto wounding resulting in a hyperplastic scar, was used. Importantly, thesame Nefopam treatment regimen, as described above, resulted in smallerscars as compared to control scars not treated with TGF-® (see FIG. 8).Thus, Nefopam is able to reduce scar size in both hyperplastic andnormal wound repair.

Example 8 Various Carriers can be Used for Topical Delivery of Nefopam

For skin wounds, an ideal product is a topical formulation of Nefopam.Topical Nefopam formulations using the following carriers were preparedand evaluated: carboxymethylcellulose (CMC), petrolatum, andhypromellose. The three carriers were tested in vivo to determine theformulation effective to deliver Nefopam through the skin. The resultsare illustrated in FIG. 9.

Example 9 Topical Nefopam Decreases Normal Scar Size in Mice

Cutaneous wounds were generated in Tcf mice using the biopsy punchprocedure described above. 6 mm diameter full thickness punch woundswere treated topically with either control cream of 1% Nefopam creamtwice daily for up to 21 days. It was observed that Nefopam treatmentresulted in a reduction in scar size by approximately one third comparedto controls. Table 1 indicates the average normal wound size (measuredin mm) in a mouse model at day 0 and following 21 days of daily topicaladministration of either 1% Nefopam cream formulated in petrolatumcarrier or carrier alone control cream. Averages are provided for 4wounds per group.

TABLE 1 Day 0 Day 21 Control Average size of wound (mm) 6 3.116666667 1%nefopam Average size of wound (mm) 6 2.02

4 mm punch wounds were generated in Tcf mice which were then treatedtopically with one of 1% Nefopam cream or control cream twice daily for14 days. The surface areas of scars formed after 14 days of treatmentwere measured using arbitrary units, where 100 arbitrary unitsrepresents the control cream treatment. Ten wounds were measured foreach treatment and data presented as mean and standard deviation. It wasobserved that scars in mice receiving Nefopam treatment weresignificantly smaller than those subjected to control treatment (p<0.05)where control treatment is 0% Nefopam (see FIG. 10).

While this invention has been described with reference to illustrativeembodiments and examples, the description is not intended to beconstrued in a limiting sense. Thus, various modification of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thisdescription. It is therefore contemplated that the appended claims willcover any such modifications or embodiments. Further, all of the claimsare hereby incorporated by reference into the description of thepreferred embodiments.

Any publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

We claim:
 1. A method of treating a dermal scar in a mammal comprisingadministering a Nefopam compound selected from Nefopam, or afunctionally equivalent, salt or solvate thereof, to the mammal, whereinthe dermal scar is not a keloid.
 2. The method of claim 1, wherein thedermal scar is selected from the group consisting of a hypertrophicscar, a hypeiplastic scar, and scars from cuts, scrapes, infection,acne, burns and surgery.
 3. The method of claim 1, wherein the Nefopamcompound is administered in combination with a pharmaceuticallyacceptable adjuvant.
 4. The method of claim 1, wherein the Nefopamcompound is administered in conjunction with an additional therapeuticagent.
 5. The method of claim 4, wherein the additional therapeuticagent is selected from the group consisting of an anti-scarring agentand a wound healing agent.
 6. The method of claim 1, wherein the Nefopamcompound is administered topically.
 7. The method of claim 6, whereinthe Nefopam compound is applied to a biocompatible matrix forapplication to a target site.
 8. The method of claim 7, wherein thebiocompatible matrix is selected from the group consisting of adressage, bandage, implant and polymeric matrix.
 9. The method of claim1, wherein the Nefopam compound is administered at a dosage of about0.0001-100 mg.
 10. The method of claim 1, wherein the amount of scartissue is reduced and/or the appearance of the scar tissue is improved.11. An method of decreasing β-catenin levels in human fibroblast cellsof a human subject having a fibroproliferative disorder comprisingadministering to the human subject an effective amount of Nefopam or apharmaceutically acceptable salt or solvate thereof, wherein β-cateninlevels in the human fibroblast cells are decreased.